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Praca oryginalna
Original paper
DOI: dx.doi.org/10.21521/mw.6449
Chronic kidney disease (CKD) is an important cause
of morbidity and mortality in dogs, and it is often a
re-sult of primary glomerular disease (8). The prevalence
of CKD increases with age, with 15% of dogs over
10 years old being affected (28). Early diagnosis may
allow therapeutic intervention that prevents further
damage and progressive decline of renal function.
However, only a decrease of > 75% of renal functional
mass will be detected by current diagnostic tests such
as blood urea nitrogen (BUN) and serum creatinine
(sCr) concentrations (10). At present symmetric
di-methylarginine (SDMA) is used as a screening tool for
early kidney dysfunction and monitoring treatment in
cases of chronic kidney disease (CKD). Unfortunately
there are no current studies describing the suitability
of this test for use with published population-based
reference intervals. It is also well know that proteinuria
is a marker and mediator of chronic kidney disease
and itself can promote further renal damage and CKD
progression. Nevertheless, the mechanism by which
excess proteins induce renal injury is still not entirely
understood (8). In clinical practice, the urinary
pro-tein-to-creatinine ratio (UP/C) and microalbuminuria
assays are of limited usefulness, because it indicates
only the magnitude of proteinuria and not the origin
of the loss (glomerular or tubular) (12). Sensitive and
specific biomarkers for early prediction and
monitor-ing of CKD in dogs have received increasmonitor-ing
atten-tion in recent years (16, 19, 29-35, 38), but they are
currently lacking. Urinary proteins of low (LMW) to
high molecular weight (HMW) have recently been
introduced and have been helpful in assessing the
localization, extent and progression of renal injuries
(21, 38). Among these biomarkers, HMW proteins
such as urinary immunoglobulin G (uIgG) are usually
associated with glomerular damage, whereas detection
of LMW proteins like retinol binding protein (uRBP)
typically reflects proximal tubular damage (1-3, 8, 38).
A marker that might be useful in the recognition of
dis-tal tubular injury is the Tamm-Horsfal protein (THP),
a glycoprotein exclusively synthesized in the cells
lining the thick ascending limb and distal convoluted
tubules (35).
The objective of this study was to assess the
localiza-tion and extent of renal damage in dogs with chronic
kidney disease using a urinary marker for glomerular
(uIgG), proximal tubular dysfunction (uRBP) and
distal tubular dysfunction (uTHP).
Utility of urinary markers in the assessment of renal
dysfunction in dogs with chronic kidney disease
DAGMARA WINIARCZYK
Department and Clinic of Animal Internal Diseases, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Głęboka 30, 20-612 Lublin, Poland
Received 02.10.2019 Accepted 30.03.2020
Winiarczyk D.
Utility of urinary markers in the assessment of renal dysfunction in dogs with chronic kidney disease
Summary
Chronic kidney disease is a common and clinically significant disease. This complication leads to a decrease of
the glomerular filtration rate and in consequence causes azotaemia and uraemia. The objective of this study was
to assess the localization and extent of renal damage in dogs with stage-3 chronic kidney disease using a urinary
marker for glomerular dysfunction, proximal tubular dysfunction and distal tubular dysfunction (uIgG, uRBP
and uTHP, respectively). The examination was performed in twelve dogs affected in stage-3 chronic kidney
disease and ten clinically healthy dogs (female and male of comparable age). The levels of urinary biomarkers
were measured by commercially available ELISA-tests. In the infected animals a significant renal excretion
of HMW protein uIgG and LMW protein uRBP was observed, indicating a dysfunction of the glomerular
and tubular regions of the kidneys. Lower levels of uTHP in dogs with CKD was noticed, which may suggest
impaired distal tubular regions of the kidneys.
Med. Weter. 2020, 76 (9), 525-528
526
Material and methods
Animals. The current study was performed at the Faculty
of Veterinary Medicine in Lublin. All owners agreed to
par-ticipate in the study and signed an informed form.
The study involved 22 mixed breeds dogs (12 males and
10 females), weighing 5-8 kg (median 6.2 kg) and aged
2-7 years (median 4.35 years), divided into two groups.
Group 1, the study group (n = 12; six males and six females),
consisted of dogs with stage –3 chronic kidney disease
(according to IRIS classification). Group 2, ten healthy dogs
(student-owned and healthy patients referred to the clinic for
vaccination purpose). Dogs were judged healthy based on
history, physical examination, hematology and biochemical
profile, and urinalysis. The diseased group was comprised
of dogs with CKD diagnosed on the basis of history,
clini-cal signs, and clinicopathologiclini-cal results. According to the
International Renal Interest Society (IRIS) CKD guidelines
all dogs in group 1 had clinical finding of CKD in 3 stage,
persistent pathological renal proteinuria based on the urine
protein to creatinine ration, assessed and confirmed over
a 2 month period (UPC > 0.5), and a serum creatinine
con-centration ≥ 2.1 mg/dl.
All dogs were submitted to a physical examination,
arte-rial blood pressure measurement (by Doppler methods, in
accordance with the guidelines American College of
Veteri-nary Internal Medicine), blood and urine sampling.
Sample collection. The clinical study involved the
col-lection of blood and urine samples. Each blood sample was
collected using a closed vacuum system to a test tube with
EDTA and subjected to haematological analysis in an Exigo
Vet analyser (Boule Sweden). The serum obtained after
centrifuging at 3000 rpm for 15 minutes at a temperature
of 4°C was analyzed in an automatic biochemical analyzer
(Mindray BS-130). The chemistry panel included: alanine
transferase, aspartate aminotransferase, total bilirubin, urea,
creatinine, glucose, albumin, and total protein. The urine
was collected from the morning midstream in containers
with protease inhibitor (20 µl per 5 ml of urine; Protease
Inhibitor Cocktail, Roche Diagnostic Corp) and divided
into portions, one of which was subjected to a complete
routine urinalysis together with sediment examination and
quantitative assessment of proteinuria using the Urine
Pro-tein to Creatinine Ratio. The UPC was measured twice, in
two samples collected at a two-week interval. UPC levels
exceeding 0.5 were considered to be proteinuria. The
spe-cific gravity was determined on the basis of measurements
with a refractometer. The remaining portion of urine was
frozen at –80°C for further analysis. The whole procedure
of sample preparation was performed within one hour from
material collection. Quantification of urinary markers uIgG,
uRBP, and uTHP was performed on thawed supernatant.
Blood pressure was measured using the Doppler method.
The measurements were made using an Ultrasonic Doppler
Flow Detector, Model 811; Parks Medical Electronics, Inc.,
Aloha, Ore. The blood pressure was measured after the
patient was acclimatized in the clinic, as the average of three
measurements. Values exceeding 160 mmHg for systolic
pressure were considered to be hypertension.
The kidney ultrasound was performed on an Esaote
Mylab machine using a microconvex 3-9 MHz transducer.
Urinary markers. All of the urine samples were
ana-lyzed using commercially available canine- or
human-specific sandwich enzyme-linked immunosorbent assays
(ELISA) (Immunology Consultants Laboratory, Newberg,
USA, MD Products North America) to determine the
con-centrations of uIgG, uRBP and uTHP. The absorbance was
measured at a wavelength of 450 nm using an ELISA plate
reader (SpectraMax M2). A 4-Parameter Logistic Non-
Linear Regression Curve-Fitting Model (MasterPlex
Soft-ware, Hitachi Solutions) was used to generate the standard
curve and calculate the concentrations of uIgG, uRBP and
uTHP. The results were normalized to urinary creatinine
concentrations (uCr) and expressed as ratios in mg/g.
Statistical analysis. The statistical analysis was
per-formed using the Mann-Whitney U test; non-parametric test
for independent samples. uIgG/uCr, uTHP/uCr, uRBP/uCr
were used as independent variables. Variables were added
one by one (forward step) and the model refitted until the
p-values were statistically significant (p < 0.05). The
statisti-cal analyses were performed using Statistica 10.0 software
(StatSoft Poland).
Results and discussion
The main pathological finding in hematology
analy-sis in group 1 was anemia (6/12), biochemistry analyanaly-sis
revealed azotemia (the average level of creatinine was
4.3 mg/dl) (Tab. 1). Almost all dogs with CKD had
symptoms of hypertension (the average of systolic
blood pressure > 160 mmHg). Renal sonographic
examinations in group 1 revealed a decrease of
pa-renchymal thickness in both kidney and renal atrophy.
The degree of proteinuria in group 1 is presented in
Table 2. The macroscopic evaluation of urine in both
groups showed yellow sample colors. None of the dogs
of the control group had proteinuria and all parameters
in hematology, biochemical analysis and ultrasound
examination were in the physiological range (Tab. 1).
The levels of urinary biomarkers (uIgG/Cr, uTHP/Cr,
Tab. 1. Blood analysis parameters for the healthy and chronic
kidney disease dogs (expressed as median and range)
Variable Group 1(n = 12) Group 2(n = 10) Reference range Leukocyte [109/L] 12.8 (10.0-19.3) 8.5 (6.3-11.1) 6-17 Limfocyte [109/L] 2.8 (1.2-5.1) 2.1 (0.6-3.8) 1.2-5.0 Erytrocyte [109/L] 4.8 (3.77-6.18) 7.8 (6.0-9.4) 5.5-8.5 Hematocryt [%] 29.5 (21.1-39.3) 51.1 (40.7-61.3) 37-5 Hemoglobin [g/dL] 11.4 (8.4-14.4) 18.4 (14.2-21.3) 12-18 Plates [109/L] 224.1 (25- 342) 93 (164-339) 200-500 ALT [u/l] 111.4 (32-413) 47 (21-101) 3-50 AST [u/l] 52 (33-129) 28 (18-46) 1-37 BIL [T] 7.3 (1.2-18.5) 0.2 (0.1-0.5) ≤ 0.60 UREA [mg/dl] 393.1 (98.2-511.7) 37.7 (27.5-59.7) 20-45 CREA [mg/dl] 4.3 (3.2-5.0) 1.2 (0.6-1.7) 1.00-1.70 GLUC [mg/dl] 103.1 (89-122) 117.2 (66-136) 70-120 ALB [g/dl] 17.1 (3.3-90) 3.7 (3.3-4.5) 3.3-5.6 TP g/dl 6.8 (5.8-7.6) 6.7 (2.2-8.6) 5.5-7.0
Med. Weter. 2020, 76 (9), 525-528
527
and uRBP/Cr) in dogs of both groups are presented
in Table 3.
Immunoglobulins G were undetectable in healthy
dogs. The average value of uIgG/uC in the group of
dogs with CKD was 305.1. In healthy dogs, the average
level of uRBP/uCr was 0.2 mg/g, in the diseased group
it was 15.1. The average value of uTHP/uCr in healthy
dogs was 0.26 and did not increase significantly, while
in the infected animals it dropped to 0.07.
In the present study we investigate protein excretion
in the urine of twelve dogs that were suffering from
Chronic Kidney Disease (group 1) and ten healthy
dogs (group 2). According to the IRIS grading criteria
the dogs were classified as CKD grade 3 (moderate
renal azotemia, proteinuria, hypertension). Thereby
we focused on the identification of urinary markers for
glomerular disorders (Immunoglobulin G) and tubular
dysfunction (uromodulin, retinol binding protein).
Increased uIgG level is usually an effect of increased
glomerular permeability (14). IgG is usually excreted
when the selective permeability of the glomerular
capillary wall is severely disrupted (8). In dogs with
different types of nephropathy, urine IgG level is
evalu-ated to characterize the severity of proteinuria (21, 25,
53). The elevated level of uIgG in dogs with chronic
kidney disease used in our study confirms the findings
of other researchers and clearly showed damage in the
course of glomerular disease.
The Tamm Horsfall protein (uromodulin) is a urinary
glycoprotein exclusively synthesized by tubular cells
in the distal part of the nephron (13, 23, 25). There are
a few small studies in veterinary medicine that have
measured the rate of urinary uromodulin excretion in
chronic disease states (29, 56). In our study the level of
uTHP/Cr in the diseased group was significantly lower
compared to the control group. It has been assumed that
the decrease in urinary THP expression reflects damage
to the thick limb of Henle’s loop and distal convoluted
tubules, or even the loss of nephrons. Therefore, low
urinary expression of THP might act as a marker of
progressive renal tubular disease.
However, further investigations in dogs are
re-quired to confirm this rationale for measuring urinary
uromodulin as a prognostic tool or as a biomarker of
kidney impairment.
uRBP was measured as a marker of proximal
tubu-lar dysfunction. In most mammals, this LMW protein
circulates in the plasma in the form of a complex with
another protein, transthyretin. Vitamin A binds this
complex and prevents RBP excretion. However, dogs
have high concentrations of transthyretin uncomplexed
RBP, filtered by the glomeruli. Under physiological
conditions, the filtered RBP is almost completely
reabsorbed by megalin-mediated endocytosis in the
proximal tubular cells, and tubular dysfunction leads
to excessive amounts of uRBP (35, 49). In our study
uRBP/Cr was significantly higher in dogs with chronic
kidney disease compared to the healthy controls.
Similar observations concerning dogs with CKD were
made by Smets et al. (37), Nabity et al. (26, 27). The
presence of RBP in the last fractions of a urine sample
may not simply be the result of saturation of the tubular
reabsorption mechanisms with MMW/HMW proteins
and their competition for receptor-binding sites (49),
but could indeed be the result of direct tubular
dam-age induced by other causes associated with CKD. In
one study the effect of inflammatory cytokines on the
culture efficiency of proximal tubular epithelial cells
was investigated. The obtained results indicated that
the exposure of tubular epithelial cells to TNFα caused
a decrease in megalin expression, the most important
receptor for the re-uptake of LMW proteins in the
renal tubules (17). Another study (3) investigating
the effect of ischemia-reperfusion injury on specific
sodium transporters on the apical membrane of the
renal tubule showed both their expression and activity
to be greatly reduced.
The results of this study suggest that all evaluated
markers for glomerular and tubular dysfunction may
improve the diagnosis and monitoring of CKD in dogs.
However further studies are needed to confirm these
result.
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Corresponding author: Dagmara Winiarczyk DVM, PhD, Department and Clinic of Internal Diseases, ul. Głęboka 30, 20-612 Lublin, Poland; e-mail: winiarczykdm@gmail.com
